1. Field of the Invention
This invention relates to a useful method of regulating expression of a desired foreign gene, in which Escherichia coli carrying a recombinant plasmid (hybrid plasmid) with an insertion of said foreign gene therein grows efficiently whereas the expression of said foreign gene is efficiently suppressed. This invention also relates to a process of producing a foreign gene product by using said regulating method in which the period of the growth of E. coli carrying the hybrid plasmid therein is separated from the period of the expression of the foreign gene.
2. Description of the Prior Art
Currently, in virtue of the progress of gene recombination technology, a method has been developed for producing a desired foreign polypeptide in a host bacterium, in which a hybrid plasmid is constructed by inserting a structure gene, which originates from the animals, plants or microorganisms and encodes for the desired foreign polypeptide, into an expression vector which permits the expression of the foreign gene in the host bacterium and then the host bacterium is cultured to produce the desired foreign gene product in large amounts.
This technology has almost established the measures for producing useful substances, such as human insulin and human growth hormones.
As host bacteria for producing products of the foreign genes in such gene recombination technology, strains of Escherichia coli are widely used because their biological characteristics have been sufficiently investigated and, further they have no known pathogenicity and can easily grow in culture media having relatively simple compositions.
In general, however, the stability of the hybrid plasmid incorporated into E. coli spp. is not necessarily high so that deletion of the hybrid plasmid from E. coli or change in construction of the hybrid plasmid occurs during the period of cell growth, which results in high incidence of hybrid-plasmid-deleted cells bearing no capability in expressing foreign genes.
For example, in the industrial scale production, where mass culture is to be carried out, relatively long incubation time is generally required even in the preliminary culture to obtain a sufficient number of bacterial cells with high activity for the use in the following main bulk culture. Thus, high incidence of the deletion of hybrid plasmids from the bacterial cells as mentioned above can not be avoided. In consequence, the number of the effective foreign genes in the culture decreases as the number of the hybrid-plasmid-deleted cells increases, which results in a poor yield of the desired foreign gene product in the final culture.
In addition, cells can not efficiently grow in a usual culture accompanied with a foreign gene expression.
In view of the aforementioned problem caused by the use of the hybrid plasmid in the culture, the method of separating the period of cell growth from that of gene expression has been brought into discussion.
More precisely, the method comprises culturing a host bacterium carrying a hybrid plasmid with an insertion of a foreign gene therein under the conditions such that the expression of the foreign gene in the hybrid plasmid is suppressed until a desired number of cells be obtained and thereafter the suppression be released to permit the expression of the foreign gene and the culture be continued. In consequence, the incidence of hybrid-plasmid-deleted cells is suppressed as low as possible so that the foreign gene product are efficiently produced.
A known example of the method of controlling expression of a foreign gene as such comprises the use of an inducible-type expression promoter as the hybrid plasmid so that the expression of the foreign gene can voluntarily induced by addition of an inducer which inactivates repressors such as the P.sub.L lambda promoter/operator of the E. coli lambda phage or the trp-promoter/operator of the E. coli tryptophan operon.
Another known example of the method of controlling expression of a foreign gene comprises the use of a temperature-dependent-type vector which has a temperature-dependent replication origin (i.e., the vector starts replication only at a certain range of specified temperatures) and thus the foreign gene expression is regulated by changing the culture temperature.
However, in the case of using the inducible-type expression promoter, the production cost tends to be high since the expression inducers are expensive and furthermore, the incidence of the hybrid-plasmid-deleted bacteria cannot be sufficiently prevented. Consequently, the method cannot be applicable to the industrial scale culture.
Furthermore, in the case of using the temperature-dependent-type vector, the incidence of the hybrid-plasmid-deleted bacteria cannot be prevented effectively and additionally, complicated apparatus for temperature control is required.